8DEN

Heme-Free Cytochrome Variant ApoCyt

Summary for 8DEN

• Entry DOI: 10.2210/pdb8den/pdb
• Descriptor: Soluble cytochrome b562 (2 entities in total)
• Functional Keywords: apo cytochrome b562, engineered protein, de novo protein
• Biological source: Escherichia coli BL21(DE3)
• Total number of polymer chains: 4
• Total formula weight: 47577.18

Authors

Hoffnagle, A.H.,Eng, V.H.,Tezcan, F.A. (deposition date: 2022-06-20, release date: 2022-07-06, Last modification date: 2023-10-18)

Primary citation

Hoffnagle, A.M.,Eng, V.H.,Markel, U.,Tezcan, F.A.
Computationally Guided Redesign of a Heme-free Cytochrome with Native-like Structure and Stability.
Biochemistry, 61:2063-2072, 2022

PubMed Abstract: Metals can play key roles in stabilizing protein structures, but ensuring their proper incorporation is a challenge when a metalloprotein is overexpressed in a non-native cellular environment. Here, we have used computational protein design tools to redesign cytochrome (cyt ), which relies on the binding of its heme cofactor to achieve its proper fold, into a stable, heme-free protein. The resulting protein, ApoCyt, features only four mutations and no metal-ligand or covalent bonds, yet displays improved stability over cyt . Mutagenesis studies and X-ray crystal structures reveal that the increase in stability is due to the computationally prescribed mutations, which stabilize the protein fold through a combination of hydrophobic packing interactions, hydrogen bonds, and cation-π interactions. Upon installation of the relevant mutations, ApoCyt is capable of assembling into previously reported, cytochrome-based trimeric and tetrameric assemblies, demonstrating that ApoCyt retains the structure and assembly properties of cyt . The successful design of ApoCyt therefore enables further functional diversification of cytochrome-based assemblies and demonstrates that structural metal cofactors can be replaced by a small number of well-designed, non-covalent interactions.

PubMed: 36106943
DOI: 10.1021/acs.biochem.2c00369

Experimental method

X-RAY DIFFRACTION (1.69 Å)